Adult stem cells properties in terms of commitment, aging and biological safety of grit-blasted and Acid-etched ti dental implants surfaces.

Titanium (Ti) is one of the most widely used biomaterials for manufacturing dental implants. The implant surface properties strongly influence osseointegration. The aim of the present study was to in vitro investigate the characteristics of Ti dental implants in terms of mutagenicity, hemocompatibility, biocompatibility, osteoinductivity and biological safety. The Ames test was used to test the mutagenicity of the Ti dental implants, and the hemolysis assay for evaluating their hemocompatibility. Human adipose - derived stem cells (ADSCs) were then seeded onto these implants in order to evaluate their cytotoxicity. Gene expression analyzing with real-time PCR was carried out to investigate the osteoinductivity of the biomaterials. Finally, the genetic stability of the cells cultured onto dental implants was determined by karyotyping. Our results demonstrated that Ti dental implants are not mutagenic, do not cause hemolysis, and are biocompatible. The MTT assay revealed that ADSCs, seeded on Ti dental implants, proliferate up to 30 days in culture. Moreover, ADSCs loaded on Ti dental implants show a substantial expression of some osteoblast specific markers, such as COL1A1, OPN, ALPL, and RUNX2, as well as chromosomal stability after 30 days of culture in a medium without osteogenic factors. In conclusion, the grit-blasted and acid-etched treatment seems to favor the adhesion and proliferation of ADSCs and improve the osteoinductivity of Ti dental implant surfaces.

The clinical success of Ti dental implants is their osseointegration, which is the formation of a strong connection between the implant surface and the surrounding host bone (6,7). It is now well documented that the surface properties of Ti implants, such as wettability, charge, chemistry and topography, are the most influencing factors in the establishment of cell-biomaterial contacts and in the improvement of osseointegration (8)(9)(10)(11). In particular, cell attachment, proliferation and differentiation into an osteoblastic phenotype seem to be strongly regulated by the surface roughness of dental implants (12)(13)(14). Plasma-spray coatings, grit-blasting, acid-etching, electrochemical processes or a combination of them are the most frequently used techniques to obtain Ti rough surfaces (15,16). Grit-blasting is usually achieved by treating the implant surface with hard ceramic, such as alumina, titanium oxide and calcium phosphate particles (17)(18)(19). Various sizes of these ceramic particles generate different roughness on Ti implants surfaces. Another method for obtaining rough surfaces consists in treating Ti dental implants with strong acids, such as HCl, H 2 SO 4 , HNO 3 and HF (20). This chemical process, known as acid-etching, improves the osteoconductive properties of implants enhancing osteoblasts adhesion, thus resulting in bone formation directly on the surface of the implant (21). However, the effects of acid-etching on the long-term stability of the Ti dental implant are rather limited. Indeed, the acid-etching technique causes hydrogen embrittlement, which leads to microcracks on the surface of the titanium dental implant. Such cracks compromise the good mechanical properties, especially fatigue resistance, of the Ti implant (22).
To avoid this drawback, acid-etching is used in combination with grit-blasting: the result is an implant surface both macrotopographically wavy and rough at the microlevel (23). In vitro and in vivo studies demonstrated that grit-blasted and acidetched surfaces show great biomechanical stability, high mechanical resistance, low risk of clinical failures, and high bond between implant and bone (24,25).
Although research is investing significantly on developing new Ti modified surfaces, a detailed understanding of the molecular and cellular mechanisms of osseointegration is still lacking.
Traditionally, bone regeneration around Ti dental implants is considered a process comparable to healing after a fracture (26). The healing process always occurs through a series of three overlapping events: inflammation, proliferation, and remodeling (27). In all these events, an important role is carried out by mesenchymal stem cells (MSCs), which have self-renewal capacity and multi-lineage potential. For example, MSCs are able to differentiate into osteoblasts, which are the cells responsible of bone growth (28). In the presence of an implant, it is crucial that these cells adhere to the dental implant surface in order to develop a bonespecific extracellular matrix (ECM), which later mineralizes to form an integrated bone-implant interface (23).

Hemolysis assay
The blood compatibility of Ti implants was evaluated by the hemolysis assay performed following standard practices set forth in ASTM F756. Blood was obtained from three healthy New Zealand rabbits, pooled, then diluted in PBS to a total hemoglobin concentration of 10± 1 mg/ ml. The implant was considered as non-hemolytic if the HI was 2% or less.

Cells seeding onto Ti implants
ADSCs at p4 were seeded onto the Ti implants at a density of 2x 10 6 cells/ implant in a 12-well plate. The cells were cultured in cDMEM without any osteogenic differentiation factor at 37 °C with 5% CO 2 up to 30 days, and the medium was

Real-time PCR
Human primers were selected for each target gene with Primer 3 software (Table 1)

Evaluation of the mutagenicity of Ti dental implants
The Ames test was performed in order to assess the mutagenic potential of Ti implants. Four different histidine dependent mutant strains (TA1535, TA1537, TA98 and TA100) of Salmonella typhimurium were used. As reported in table 2, no mutagenic activity has been revealed.

Evaluation of the hemocompatibility of Ti discs
The hemolysis assay was performed in order to evaluate the blood compatibility of the Ti implants, which are intended for blood contacting applications. The HI was less than 2%, indicating the absence of any hemolytic activity of the tested material ( Table 3).

Biocompatibility of Ti implants
In order to evaluate the biocompatibility of Ti implants, ADSCs were seeded and cultivated onto these surfaces up to 30 days. The results of MTT assay show that the cells were able to adhere and proliferate onto the Ti implants (Fig. 1).

Expression of osteoblast markers
The

Cytogenetic analysis
The chromosomal stability of ADSCs seeded on the Ti implants was analyzed by means of karyotyping. As reported in figure 4, no chromosomal alterations are present in ADSCs seeded onto these surfaces for 30 days.

Discussion
Ti and its alloys are the most commonly used biomaterials in dental implantology. Nevertheless, a question that remains to be answered is how